Virus production

ABSTRACT

An improved process for recovery of virus from allantoic fluid of virus-infected chick embryos. Virus associated with granular and fibrous debris in the allantoic fluid can be disassociated from the debris and recovered, thereby increasing viral yield. Dissociation can be achieved by subjecting the virus-debris complex to conditions of increased salt concentrations, e.g., 0.5 M or greater.

This application claims priority to U.S. provisional application Ser.No. 60/479,723, filed Jun. 20, 2003, U.S. provisional application Ser.No. 60/540,782, filed Jan. 30, 2004, and U.S. provisional applicationSer. No. 60/572,718, filed May 20, 2004, all of which are incorporatedby reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to the recovery of enveloped virus fromallantoic fluid of virus-infected chick embryos. The heightenedrecoveries facilitate production of viral vaccines, especially influenzavaccine, and can also provide enhanced yields of viral proteins,including heterologous proteins expressed by viral vectors.

BACKGROUND OF THE INVENTION

Upon infection by a pathogen, the host's immune system recognizesantigens on or in the pathogen and directs an immune response againstthe antigen-containing pathogen. During this response, there is anincrease in the number of immune cells specific to the antigens of thepathogen and some of these cells remain after the infection subsides.The presence of the remaining cells prevents the pathogen fromestablishing infection when the host is subjected to the pathogen at alater time. This is referred to as protective immunity.

Vaccines provide protective immunity against pathogens by presenting apathogen's antigens to the immune system without causing disease.Several methods have been developed to allow presentation of antigenswithout disease-causing infection by the pathogen. These include using alive but attenuated pathogen, an inactivated pathogen, or a fragment(subunit) of the pathogen.

Because therapy for many viral infections remains elusive, it ispreferred to prevent or moderate infection through vaccination ratherthan treat the infection after it occurs. Examples of particularlyproblematic infectious viruses are those of the orthomyxoviridae,especially influenza virus, paramyxoviridae, flaviviridae, togaviridae,rhabdoviridae, and coronaviridae families. Millions of people arevaccinated against one or more members of these virus families eachyear.

While some viruses will propagate well in cell culture, others requirepropagation in embryonated chicken eggs with virus recovery fromallantoic fluids. Influenza vaccine has been supplied to the populacefor many years as a multi-strain combination product recovered from theallantoic fluids of embryonated chicken eggs. Three strains, selectedannually from a large panel of strains, are grown, purified, and pooledto create a given vaccine. The growth of each selected strain ofinfluenza can vary markedly, often leading to difficulties inefficiently meeting the annual market demand for such a trivalentvaccine.

Various methods have been proposed to improve and/or simplify therecovery of virus or viral products from feedstock. U.S. Pat. No.3,627,873 describes a process in which virus is extracted fromconcentrated allantoic fluid feedstock using diethyl ether andmethylacetate. Still further yield improvements are said to have beenobtained using multiple extractions with both butyl and ethylacetatesaccording to U.S. Pat. No. 4,000,257.

U.S. Pat. No. 3,316,153 describes a multi-step extraction process, aimedat separating virus particles from cellular debris and is assertedlyapplicable to feedstocks that derive from virus-infected chick allantoicfluid or from cell or tissue-culture fluids. In this method, virusadsorbed to precipitated calcium phosphate is dispersed in EDTA at pH7.8-8.3, causing dissociation and an EDTA-based sequestering of thesoluble calcium, thereby releasing the virus for recovery. The resultingvirus-containing solution is dialyzed against water or preferably anaqueous glycine-sodium chloride solution to reduce the EDTA andphosphate content.

U.S. Pat. No. 4,724,210 describes methods for purification of influenzausing ion exchange chromatography. An influenza-containing solution,e.g. allantoic fluid, is passed through cellulose sulfate column whereinthe virus is adhered to the column packing. The column is subsequentlywashed and virus eluted with a solution containing 1.0 M to 1.5 M sodiumchloride. This is followed by a 4.99 M sodium chloride wash.

In WO 02/067983, preparation of a split influenza vaccine is describedas involving moderate-speed centrifugation to clarify allantoic fluid,adsorption of the clarified fluid on a CaHPO₄ gel, followed byresolublization with an EDTA-Na₂ solution. See also WO 02/08749describing the same process.

In U.S. Pat. No. 4,327,182, allantoic fluid feedstocks from the growthof influenza virus are subjected to a multi-stage extraction processaimed at recovering influenza subunits, haemagglutinin (HA) andneuraminidase (NA). The technique relies on a concentration step inwhich virus feedstock is present with detergent and a saline solutionfollowed by successive filtration to remove non-viral particles.

U.S. Pat. No. 3,962,421 describes a method for the disruption ofinfluenza viruses. Allantoic fluid is subjected to high-speedcentrifugation. The resulting pellet is resuspended in saline andball-milled for 12-15 hours to create a virus suspension. The virussuspension is then treated with phosphate-ester to disrupt the virusparticles into lipid-free particles (subunits) that carry the surfaceantigens of intact viruses.

In U.S. Pat. No. 3,874,999, allantoic fluids containing influenza virusare centrifuged at low speeds to remove gross particles. The virus isthen removed from the supernatant by high-speed centrifugation andresuspended in a phosphate buffer. Nonvirus proteins and lipids areremoved by treatment of the suspension with 0.1-0.4 M magnesium sulfateat an alkaline pH for 16-18 hours at 4° C. The resultant suspension isclarified by low speed centrifugation and the virus is purified from theresulting supernatant.

Of particular interest to the background of the invention are viralrecovery manipulations involving the contact of non-allantoic fluidvirus sources with solutions having elevated concentrations of one ormore salts and studies of the effect of various salt concentrations onpurified virus.

Some processes assertedly provide for increased yields or greater purityof virus when infected cells are contacted or incubated with solutioncontaining elevated salt concentrations followed by purification of thevirus from the solution.

In WO 99/07834, herpesvirus infected Vero cell cultures are incubated ina hypertonic aqueous salt solution (e.g., 0.8 to 0.9 M NaCl) for severalhours. The solution is then removed and herpesvirus harvested from thesolution. This method was asserted to be superior to methods wherein thecells are subjected to ultrasonic disruption.

Others have addressed contacting virus-infected cultured cells withelevated salt concentrations.

U.S. Pat. No. 5,506,129 reports increased yields of hepatitis A virusafter growing infected BS-C-1 cells in growth medium containing ˜0.3 MNaCl.

Karakuyumchan et al. (Acta virol.:155-158, 1981) reports that rabiesvirus obtained after shaking infected brain tissue in a 0.3 M NaClcontaining buffered solution lacks neuroallergenic activity caused byresidual brain tissue.

Pauli and Ludwig (Virus Research, 2:29-33, 1985) reports increasedyields of Boma disease virus from a virus-infected cell lines grown inmedium containing ˜0.3 M NaCl.

Various groups have studied the effect of contacting purified viruseswith elevated salt concentrations on the characteristics of the virus.

In Breschkin et al. (Virology, 80:441-444, 1977), a particular mutatedmeasles virus lacking hemagglutination activity in isotonic saline haswild-type level hemagglutination activity in 0.8 M (NH₄)₂SO₄, whereasthe high salt has no effect on the hemagglutination activity of awild-type virus.

Wallis and Melnick (Virology, 16:504-506, 1962) report that, while highsalt (1 M MgCl₂, 1 M CaCl₂, or 2 M NaCl) prevents heat inactivation ofpolio, coxsackie, and ECHO viruses, 1 M MgCl₂ enhances inactivation ofadeno-, papova-, herpes-, myxo-, arbor, and poxviruses.

In Willkommen et al. (Acta virol., 27:407-411, 1983), purifiedlyophilized influenza virus is reconstituted in buffered salinecontaining increasing concentrations of NaCl (up to 1.15 M).Subsequently, the reconstituted virus is cleaved with detergent and asingle-radial-immunodiffusion (SRD) test performed. With some strains ofinfluenza virus, increasing the salt concentration in the reconstitutionbuffer shows no effect on the results of an SRD test to hemaggluinin(HA). However, other strains, when reconstituted in buffered salinecontaining 1.15 M NaCl, give a HA concentration in the SRD test that istwice that of the same strain reconstituted in buffered salinecontaining 0.15 M NaCl. The authors identify viral aggregation aspossibly blocking detergent penetration and attenuated the SRD response.

Molodkina et al. (Colloids and Surfaces A: Physicochemical andEngineering Aspects, 98:1-9, 1995) report that increasing saltconcentrations up to 0.3 M NaCl leads to dispersion of purifiedinfluenza virus aggregates.

Sudnik et al. (Vyestsi Akademii Navuk BSSR Syeryya Biyalahichnykh Navuk,6:71-77, 1985) report high ionicity can partially offset the destructionof the influenza virus envelope at pH 2.2.

Also of interest to the background of the invention are the results ofstudies by Makhov et al. (Voprosy Virusologii, 34(2):274-279, 1989) onthe proportion of filamentous influenza virions in allantoic fluids. Ina context divorced from virus recovery, Makhov et al. report thatpresence of filamentous influenza virions in allantoic fluids is strainspecific and ionic-strength dependent. Allantoic fluids were examinedusing electron microscopy to determine the presence of filamentousvirions. The occurrence of filamentous virions in the allantoic fluidfor one particular influenza strain was 7.1%. When the NaClconcentration was raised to 0.25 M or 1.0 M, the occurrence was reducedto 0.37% and 0.16%, respectively.

Thus, there remains a need in the art for an improvement of the purityand yield of viruses from allantoic fluid of virus-infected chickembryos.

SUMMARY OF THE INVENTION

The invention provides an improvement in a process for recovering virusfrom allantoic fluid of virus-infected chick embryos. As disclosedherein, a considerable portion of the virus within the allantoic fluidhas now been found to be associated with granular or fibrous debris andis therefore lost when the allantoic fluid is clarified to remove thedebris. By dissociating the virus from the debris prior to finalseparatory processing, viral yields are improved.

Processes to recover virus from allantoic fluid often contain a stepwherein the allantoic fluid is subjected to clarification, e.g., bycentrifugation or filtration, to form a clarified liquid fraction and adebris-containing fraction. When the allantoic fluid is clarified bycentrifugation, the debris-containing fraction is typically in the formof a pellet; when clarified by filtration, it is typically in the formof a retentate. By including the step of extracting virus from thisdebris-containing fraction, the invention provides an improvement overknown processes of recovering virus from allantoic fluid of infectedchick embryos.

A preferred method of extracting virus the debris-containing allantoicfluid fraction is to dissociate the virus into a suspension having anon-isotonic concentration of one or more salts. In particular, virus isreadily dissociated from the debris with solutions comprising one ormore salts having a total salt concentration therein of about 0.5 M orgreater or equivalent mole ratio of salt per ml of allantoic fluid.Particularly useful solutions are phosphate buffered solutions having apH ranging from 3 to 10 and comprising a total salt concentration (e.g.,total NaCl concentration) from about 1.0 M to about 3.5 M. Dissociatedvirus can be recovered from the suspension. Recovery can include asecond clarification forming a second clarified liquid and a seconddebris-containing fraction. Preferred methods of recovering virus alsoinclude localization of the virus on a sucrose density gradient.

In some aspects, the invention provides a process for recovering virusfrom allantoic fluid of virus-infected chick embryos by adding one ormore salts to the allantoic fluid to generate a total salt concentrationtherein of about 0.5 M or greater followed by recovering virus from theresulting fluid. The salt can be added in the form of an aqueoussolution, such as concentrated phosphate buffered saline (PBS). In oneembodiment, the salt is added to the allantoic fluid prior to removingthe allantoic fluid from an egg. After addition of the one or moresalts, recovery of the virus can include a clarification step to form adebris-containing fraction. Any virus remaining in the debris-containingfraction can be extracted to optimize viral yields. Alternatively, thesalt-treated allantoic fluid may be directly processed by, e.g., sucrosedensity gradient fractionation with or without removal of water toreduce the volume of fluid subjected to fractionation.

In a particularly preferred embodiment, a solution of one or more saltsis added to allantoic fluid to generate a total volume concentrationtherein of about 1.5 M or greater. The pH of the allantoic fluid alsocan be adjusted or maintained. Preferred ranges of pH include pH 3.0 topH 6.8 or pH 6.8 to pH 9.8. After the salts are added to the allantoicfluid, it is clarified by centrifugation or filtration. The clarifiedallantoic fluid is then subjected to sucrose density gradient separationto localize virus. Subsequently, localized virus is isolated from thegradient.

The methods of the present invention can be used to recover essentiallyany virus that replicates in virus-infected chick embryos and is presentin the allantoic fluid. Particularly preferred viruses are the envelopedRNA viruses, including members of the orthomyxoviridae, paramyxoviridae,flaviviridae, togaviridae, rhabdoviridae, and coronaviridae families. Asdemonstrated in the Examples, methods of the present invention improvethe recovery of both Influenza A and Influenza B virus considerably.

Other features and advantages of the invention will become apparent fromthe following detailed description. It should be understood, however,that the detailed description and the specific examples, whileindicating preferred embodiments of the invention, are given by way ofillustration only, because various changes and modifications within thespirit and scope of the invention will become apparent to those skilledin the art from this detailed description.

DESCRIPTION OF THE DRAWING

FIG. 1. Treatment of pellets from clarified allantoic fluid with asolution containing 1.6 M NaCl increases yield and provides betterlocalization in a sucrose gradient.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In one aspect, the invention provides an improved process for recoveryof virus from allantoic fluid of virus-infected chick embryos. Thisprocess significantly improves the yield of virus from allantoic fluidand provides highly purified virus compositions, or derivative virussubunit preparations, useful to prepare vaccines.

As used herein and in the claims, “virus” shall mean enveloped,preferably intact and infectious, viral particles as opposed to viralfragments, components and/or individual viral antigens such as obtainedby well-know splitting techniques. Following recovery of virus accordingto the present invention, viral particles may readily be subjected tofragmentation or splitting.

Processes of the invention are applicable to both naturally occurringviruses and genetically modified viruses, such as those described, forexample, in WO 99/66045 and its counterpart publication US 2003/0087417.

In a preferred embodiment, virally infected allantoic fluid from chickembryos is prepared according to guidelines currently established forvaccine production. Generally, this process entails the use of 9-12 dayold embryonated chicken eggs that are pre-candled to eliminate spoiledor unfertilized eggs. The remaining eggs are then inoculated in theamniotic and/or allantoic cavity with the particular strain of livevirus for which a vaccine is desired. The eggs are incubated at 32-37°C. typically for two or three days, post-candled to eliminate spoiledeggs, and the eggs are next refrigerated at a temperature of about 4-6°C. for about 24 hours before the egg fluids are aseptically harvested.The allantoic fluid so harvested contains a high concentration of livevirus. This process is useful particularly for the production ofinfluenza virus of various types including most or all strainsinfluenza-A and influenza-B.

As demonstrated in Example 1 below, although the virus infectedallantoic fluid contains a high concentration of live virus, much of thevirus is associated (aggregated) with fibrous or granular debris and islost when the debris is typically separated from the allantoic fluid byclarification. By employing elevated salt concentrations to dissociatevirus from the debris and recovering the dissociated virus, the methodsof the present invention provide increased viral yields from allantoicfluid. Virus can be dissociated from the debris within the allantoicfluid (either within the infected egg or after the allantoic fluid isremoved from the egg) prior to any clarification. Indeed, in someinstances, clarification can be dispensed with as a preliminary recoverystep prior to, e.g., sucrose density gradient separation. Alternatively,the allantoic fluid can be clarified to form a debris-containingfraction and the virus can be subsequently dissociated from the debrisin this debris-containing fraction. After dissociation from the debris,virus can be recovered using conventional virus purification techniquesas described below.

A preferred method of dissociating virus from the aggregated debris isto place the virus associated with the debris in an environment having anon-isotonic salt concentration. The environment is said to have a“non-isotonic” salt concentration when it differs significantly fromthat of allantoic fluid, which has a total salt concentration of about150 mM. Examples of non-isotonic salt concentrations include, but arenot limited to, 10 mM or less, 20 mM or less, 30 mM or less, 40 mM orless, 50 mM or less, 60 mM or less, 70 mM or less, 80 mM or less, 90 mMor less, 100 mM or less, 110 mM or less, 120 mM or less, 130 mM or less,140 mM or less, which concentrations can result from dilution ofallantoic fluid with water. Dilution with isotonic salt solutions suchas phosphate buffered saline will not render the allantoic fluidnon-isotonic but, as noted below, may have beneficial effects in termsof dissociating virus from debris.

Non-isotonic salt concentrations includes hypertonic salt concentrationssuch as 160 mM or greater, 170 mM or greater, 180 mM or greater, 190 mMor greater, 0.2 M or greater, 0.3 M or greater, 0.4 M or greater, 0.5 Mor greater, 0.6 M or greater, 0.7 M or greater, 0.8 M or greater, 0.9 Mor greater, 1.0 M or greater, 1.1 M or greater, 1.2 M or greater, 1.3 Mor greater, 1.4 M or greater, 1.5 M or greater, 1.6 M or greater, 1.7 Mor greater, 1.8 M or greater, 1.9 M or greater, 2.0 M or greater, 2.5 Mor greater, 3.0 M or greater, and 3.5 M or greater and may be obtainedby direct addition of free salt or preferably, by addition ofconcentrated salt solutions.

In all embodiments, one or more salts are added to the allantoic fluidto accomplish dissociation of virus from the aggregated debris. Oncevirus dissociation occurs, the virus-containing solution could bediluted, e.g., rendered more isotonic (i.e. less hypertonic) again,prior to recovering the virus. Alternatively, the allantoic fluid can bediluted prior to or concurrently with salt addition and the dilutedsolution may thereafter be concentrated to increase the saltconcentration thereby dissociating virus from the aggregated debris,prior to recovering the virus. In such embodiments, a preferred moleratio of salt to original volume of allantoic fluid is created.

For example, in illustrative example 10 below, a 100 ml aliquot ofallantoic fluid was diluted by the addition of 50 ml of 1×PBS, bringingthe sample volume to 150 ml. An equal volume (150 ml) of 20×PBS wassubsequently added to the sample to create a final volume of 300 ml.Allantoic fluid and 1×PBS have a NaCl concentration of about 0.15 M (150mM). Thus, there was 0.015 moles NaCl (0.1 L×150 mM NaCl) in theoriginal allantoic fluid. Then 0.0075 moles NaCl (0.05 L×150 mM NaCl)was added by the dilution with 1×PBS. The addition of the 20×PBS added0.45 moles NaCl (0.15 L×3.0 M NaCl). The 300 ml final volume contained0.4725 moles of NaCl (0.015+0.0075+0.45). Therefore, the mole ratio ofsalt to allantoic fluid was 0.4725 moles per 100 ml allantoic fluid or4.725 mmoles per ml of allantoic fluid starting material. Had theadjustment of allantoic fluid been to 0.5 M NaCl, the mole ratio wouldhave been 1.5 mmoles NaCl per ml of allantoic fluid starting material,regardless of the adjusted (total) volume.

Preferred salts are those that are generally regarded as safe (GRAS) foruse in human pharmaceuticals. The preferred salt is sodium chloride. Thesalt can also be formed from monovalent, divalent or multivalent cationmixtures thereof and can include or specifically exclude ammoniumsulfate. Thus, KCl, LiCl, CaCl₂, MgCl₂ and other salts are envisioned ascombinations of salts. Other salts include a variety of inorganic saltsand organic salts (e.g. sodium acetate, potassium acetate, etc.). Inembodiments wherein elevated salts concentrations are used to dissociatevirus from the aggregated debris, the salts selected for use in thepresent method should be those salts which remain soluble at the highconcentration required to confer the desired environment. Any salt thatcan substantially increase the ionicity (osmolarity) of a solution whileretaining solubility is suitable. Such salts include sodium chloride andpotassium chloride and the like.

In a preferred embodiment, virally infected allantoic fluid prepared inthe established manner is admixed with an aqueous solution containingsalt at high concentration, so that the resulting admixture contains thesalt at a molarity of at least 0.5 M and at most saturation, moredesirably at a molarity in the range from 1.0 M to 3.5 M. This canusually be achieved, for instance, by mixing equal volumes of allantoicfluid and salt solution, or using any other blending procedure thatprovides the desired salt concentration. In certain embodiments, theallantoic fluid can be removed from the egg prior to addition of thesalt solution. In other embodiments, salt solution is added to theallantoic fluid within the egg, or used to wash the allantoic chamberafter collection of the bulk allantoic fluid.

Preferably, the admixture of allantoic fluid with salt is also buffered,using for instance a phosphate buffering system (e.g., 20-250 mM) in theusual manner to provide a desired pH. A preferred pH range is from 3.0to 10.0. The pH can be adjusted to maximize the recovery on avirus-by-virus basis. Yields can further be enhanced by tailoring the pHof the concentrated salt/feedstock admixture to within a range preferredfor a given virus type or subtype. A preferred pH range is from 3.0 to10.0. For instance, Moscow strains of Influenza A provide higher yieldswhen the non-isotonic environment has a relatively neutral/slightlyacidic pH in the range from 6.8-7.1. However, under the same saltconditions, yields of certain Yamanashi strains of Influenza B aregreater when the non-isotonic environment has a higher pH level, about8.4.

The environment in which the virus is dissociated from debris canfurther comprise a divalent cation chelating agent, such as EDTA. Thechelating agent serves the purpose of sequestering divalent cations thateffect precipitation or agglomeration of virus particles, thus renderingthem either invisible to the titre measurement or unavailable to theinactivation conditions used during the vaccine production phase of theprocess. Suitable chelating agent concentrations are those that fosterseparation of agglomerated virus particles. In the case of EDTA,concentrations suitably are within the range from 1.0 mM to 1.0 M,depending on the initial divalent cation concentration. Other additives,alone or in combination, include reducing agents, such as DTT, andwetting agents, such as the nonionic detergents Triton X-100 andPluronic F68.

As noted above, in certain embodiments, one or more salts are added toallantoic fluid from virus-infected chick embryos to elevate the saltconcentration therein. In certain embodiments, this entails theadmixture of allantoic fluid and a concentrated salt solution that isoptionally pH adjusted and optionally contains a chelating agent, atroom temperature (18-25° C.) for convenience. The admixture can bestirred. The admixture is then chilled to preserve virus infectivitywithout precipitating the salt and the virus-containing suspension isformed either during a non-agitation resting period or, desirably, bycentrifugation or filtration.

The virus-containing supernatant or filtrate is then processed asdesired to further enrich for virus. Typically, the supernatant is nextsubjected, in the manner established for raw allantoic fluid, to asucrose gradient centrifugation processing that localizes virus on orwithin the gradient. The localized virus can be recovered from thegradient to yield virus-containing fractions. One or more pooledvirus-containing fractions can be obtained following the sucrose-basedseparation process, to form an enriched virus extract. This enrichedvirus extract can also be subjected to the high salt treatment,preventing or removing aggregates of virus that are commonly induced bygradient purification.

It is to be appreciated that the present method is not limited to anyparticular fractionation or separation process, but instead can beapplied with any other fractionation or separation processes useful toobtain purified virus from allantoic fluids, including those utilizingsize exclusion chromatography, centrifugation, filtration, solventextraction, ion-exhange chromatography, and the like. Moreover, any oneor more of the resulting fractions can be rendered non-isotonic, toimprove the virus recovery process, in that virus is renderedsubstantially monodisperse in solution.

In certain embodiments, the recovered virus is inactivated. Theinactivation process can be any of those already established in vaccineproduction, including formalin fixation, irradiation, detergent orsolvent splitting and the like.

The present invention can be applied for the recovery and purificationof a wide range of viruses. In preferred embodiments, the method isapplied for the recovery of an enveloped virus comprising an RNA genome.Such viruses include those of the family orthomyxoviradae (e.g.,influenza viruses), paramnyxoviridae (e.g., mumps virus, Sendai virus,and Newcastle disease virus), flaviviridae (e.g., Japanese encephalitisvirus and yellow fever virus), togaviridae (e.g., rubella),rhabdoviridae (e.g., vesicular stomatitis virus and rabies virus), andcoronaviridae (e.g., avian infectious bronchitis virus). In particularembodiments, the method is applied for the recovery of influenza virus,including strains of Influenza A, Influenza B, Influenza C, avianinfluenza virus, equine influenza virus, and swine influenza virus.

As noted above, in certain practices of the invention, the allantoicfluid is diluted prior to recovery of virus. In other aspects, theresulting debris-containing fraction of an initial clarification isresuspended, treated with high salt to liberate virus from the debris,reclarified, and the resulting clarified solution added back to theoriginal clarified solution. Thus, the volume of virus-containingsolution may increase during processing.

Working with large volumes of virus-containing solution may becumbersome, particularly when recovering virus from sucrose densitygradients. Methods of reducing the volume of virus-containing solutionswithout significant loss of virus are known in the art. For example, thevirus-containing solution may be subjected to tangential flow filtration(TFF) or diafiltration. In TFF, viruses in solution are passed throughhollow fiber filter tubes or across plates of filter material. Asopposed to normal flow filtration wherein the feed flow and pressure arein the same direction, TFF relies upon pressure that is perpendicular tothe feed flow. Thus, in TFF, the filtrate passes through themembrane-containing walls of the tube while the retentate flows down thepath of the tube. During this process, solution volume can be reduced asdesired.

In certain embodiments, it may be desirable to subject avirus-containing solution to diafiltration. During diafiltrationsurfactants, proteins, or other solutes that freely permeate themembrane are removed from the solution. Generally, there are two commonmodes of diafiltration: Batch and constant-volume. During batchdiafiltration, a large volume of buffer or solution is added and thenthe retentate is concentrated. During constant-volume diafiltration,buffer or solution is added at the same rate that the filtrate isremoved.

Membranes for use in TFF or diafiltration of virus-containing solutionsare commercially available (e.g., MILLIPORE, Billerica, Mass.). Inpreferred embodiments, the membrane cutoff range is 100 kD -0.05 μm.

In certain embodiments, the present invention can be applied to thepurification of one or more proteins encoded by a virus and, in someinstances, secreted into allantoic fluid by infected embryonic cells.The protein can be a viral protein or a protein encoded by aheterologous gene contained in a recombinant virus vector. In preferredembodiments, one or more salts are added to the allantoic fluidcontaining the protein to dissociate the protein from the debris.Alternatively, the protein associated with fibrous debris is separatedfrom the allantoic fluid, e.g., by centrifugation or filtration, and thedebris-containing fraction is then subjected to a non-isotonic saltconcentration to dissociate the protein from the debris. The dissociatedprotein is subsequently purified using standard techniques.

When applied to the recovery of influenza viruses from chick allantoicfluid, the present invention provides a significantly reduction of thenumber of eggs that are required for a given influenza vaccineproduction run. This, in turn, reduces the possibility of vaccineshortages at the start of the annual flu season. Sundry benefits alsoinclude a smaller total workload and an easing of waste managementissues. All of these benefits significantly reduce the cost of producinginfluenza vaccines.

In specific embodiments of the invention as applied to the extraction ofinfluenza from allantoic fluids, the present method can be applied inthe following particular manner.

Infected allantoic fluids containing high titre influenza virus vaccinestrains are harvested under standard industry procedures and eitherprocessed immediately or stored in a frozen state (e.g., at −70° C.)prior to further processing.

When processed, liquid allantoic fluid is treated with an equal volumeof phosphate-buffered 16-20% (w/v) NaCl at a pH usually in the range 6.5to 8.5, depending on the strain of influenza virus to be purified, andwith or without a chelating agent such as EDTA or other additives. Aftera suitable incubation, approximately 5 minutes or longer, where virushas dissociated from fibrous debris within the allantoic material, viruscan be purified from the solution by sucrose density centrifugation.Alternatively, one or more clarification steps may be employed. Incertain embodiments, the viral preparation is clarified bycentrifugation at up to 14,000×g, e.g., 2,000-5,000×g. The supernatantis significantly enriched for live virus over supernatants not receivingthe high-salt treatment. Pelleted debris may optionally be treated witha solution containing a high concentration of salt to extract any viruswhich may not have been liberated from the contaminating debris.

Alternately, the allantoic fluid may be clarified to form a clarifiedliquid fraction and a debris-containing fraction. Preferred methods ofclarification include centrifugation and filtration. The resultingpellet or filter retentate is suspended or washed in high saltconcentration solutions to liberate virus, which optionally can bepooled back into the bulk allantoic fluid with or without clarificationby centrifugation or other means.

The clarity of the treated supernatant can generally facilitate furtherpurification by sucrose density centrifugation. A step or continuous30-50% (w/v) sucrose gradient is typically employed. Manufacturers ofinfluenza vaccine typically use continuous flow centrifugationstrategies.

Finally, live influenza virus retrieved from sucrose gradients usuallyhas a significant proportion of the virus in an aggregated state.Further treatment of the isolated gradient fractions or fraction poolswith phosphate-buffered high salt solution, or other solution of highionic strength, at a pH usually in the range 6.5 to 8.5, liberatesaggregated live influenza virus and maximizes virus yield, which can bemonitored by HA titre, infectivity assay, immunoassay and electronmicroscopy.

The post-purification treatment of virus preparations achieves asolution of disaggregated or, ideally, monodisperse virus particles,which can be further manipulated with less loss than typicallyencountered. For example, formalin inactivation of gradient-purifiedvirus is often not completely effective, due to the presence of virusaggregates, and leads to significant product loss and necessitatespost-formalin processing. In the case of influenza vaccines, thepresence of live virus following formalin treatment requires theapplication of ether extraction or other manipulation. After high-salttreatment, formalin treatment of the virus preparation is less likely tofail, and product loss due to aggregation is greatly reduced.

A further benefit of the methods of the present invention is thatdissociation of virus from debris in the allantoic fluid leads torecovery of virus stocks having increased purity, i.e, containingsignificantly less contamination by egg components. Because eggcomponents, such as ovalbumin, can cause an allergic reaction in certainindividuals, the methods of the present invention are thought to providefor vaccines having a greater purity and, thus, have a decreasedlikelihood in causing an allergic reaction in an individual receivingthe vaccine. For example, one or more sucrose gradient fractionations ofinfluenza virus which has been subjected to dissociation from allantoicfluid debris by elevated salt treatment will ordinarily be sufficient toprovide products having no detectable ovalbumin.

Embodiments of the present invention will be described with reference tothe following examples, which are presented for illustrative purposesonly and are not intended to limit the scope of the invention.

EXAMPLES Example 1 In Influenza-Infected Allantoic Fluids, Most Virus isPresent in Highly Insoluble Virus/Debris Aggregates

Raw allantoic pools were assayed by HA (End-point determination) withand without clarification by centrifugation (Eppendorf microcentrifuge,5,000 RPM for 5 minutes).

TABLE 1 Titre (HAU/mL) Virus Strain Untreated Clarified Flu A/NewCaledonia 2,560 640 Flu A/Panama 1,280 320 Flu A/Moscow 1,280 320 FluA/Texas 10,240 320 Flu B/Yamanashi 2,560 640 Flu B/Hong Kong 1,280 160

Table 1 indicates that clarification by centrifugation typically causeda four-fold reduction in HA titer, although far higher increments wererecorded (Flu A/Texas). Overall, the data show the majority of influenzapresent in allantoic pools is in a low solubility form, easily removedby physical manipulation.

Example 2 Treatment of Allantoic Fluids Increases Soluble InfluenzaVirus Titre

80 μL aliquots of virus of crude Influenza A/Moscow-infected allantoicfluid were mixed with 80 μL 3 M NaCl solution. Salt-treated virussamples and controls were incubated 15 min on ice with occasionalmixing, then clarified by centrifugation (Eppendorf Microfuge, fullspeed, 30 seconds).

HA Assay: A 100 μL sample of each supernatant specimen was serially(2-fold) diluted by transfer of 50 μl into wells containing 50 μlphosphate buffered saline (PBS). An equal volume (50 μl) of 0.5% (v/v)chick RBC suspension was added, mixed, and allowed to settle at roomtemperature (1-2 hr). End point HA titre was determined for each testsample as the final well in the dilution series in which completehemagglutination was observed.

Typically, as indicated in Table 2, a four-fold increase in HA titre wasseen in salt-treated Flu A/Moscow infected allantoic fluid samplesversus controls.

TABLE 2 HA End-point Titres (HA units/mL) 1.6 M NaCl SAMPLE 0.15 M NaCl(final) Flu A/Moscow 5.1 2,560 10,240 Flu A/Moscow 5.3 320 2,560 FluA/Moscow 11.1 1,280 10,240 Flu A/Moscow 11.2 2,560 10,240

Example 3 Treatment of Allantoic Debris Liberates Soluble InfluenzaVirus

1.5 M NaCl was applied to Flu A and B pools. Control samples weretreated with 0.15 M NaCl, and each preparation was centrifuged forvarying times to assess the amount of virus partitioning in thesupernatant versus the pellet.

Each virus sample was aliquoted (2×300 μl) and mixed with 1 volume of 3M NaCl or 0.15 M NaCl (control). Samples were mixed and aliquoted into6×100 μl. After a 30 min incubation, samples were centrifuged at 10,000RPM for 0, 2, 4, or 6 minutes (Eppendorf Microcentrifuge), and the 100μl supernatants were retrieved and transferred to HA assay plates.Pellets were resuspended in 100 μl of 1.6 M NaCl, centrifuged for 2minutes, and the pellet washes were transferred to HA assay plates.

Results are summarized in Tables 3 and 4. Values are HA endpointsexpressed in HA Units/mL.

TABLE 3 Flu B/Yamanashi Centrifugation 1.6 M NaCl 0.15 M NaCl TimeSupernatant Pellet Supernatant Pellet 0 2560 — 2560 — 2 1280 1280 0 25604 1280 640 0 5120 6 1280 640 0 5120

TABLE 4 Flu A/Moscow Centrifugation 1.6 M NaCl 0.15 M NaCl TimeSupernatant Pellet Supernatant Pellet 0 2560 — 1280 — 2 2560 320 6401280 4 2560 640 640 1280 6 2560 640 640 1280

Example 4 Treatment Greatly Increases Virus Yield in SucroseGradient-Purified Influenza a Virus

Allantoic fluid samples for high salt treatment were clarified bycentrifugation, and virus was retrieved from the debris pellet by two10% volume 1.6 M salt washes (overnight, and then 1 hour). Washes werereclarified and then pooled back with the allantoic supernatants.

Control samples were clarified using a coarse glass fibre ‘depth’filter, to mimic the typical process used for vaccine manufacture.Controls remained slightly cloudy after filtration. Secondaryfiltration, through 1 μm or 0.45 μm filters, was not employed, therebyproviding a worst-case-scenario for technology-based yield gains.

Each of the allantoic preparations was fractionated on 6 mL sucrose stepgradients to achieve a 17:1 loading ratio. Allantoic samples were loadedonto gradients in several steps to achieve the overall loading ratio.

TABLE 5 Flu A/New Caledonia Gradient Loading Experiment Salt Treated HAUControl HAU Recovery Ratio 1 5,232,640 86,816 60:1 2 4,149,120 95,48844:1

TABLE 6 Flu A/Moscow Gradient Loading Experiment Salt Treated HAUControl HAU Recovery Ratio 1 368,320 8,944 40:1 2 591,200 9,956 60:1 3223,232 7,476 30:1 4 289,984 6,516 44:1

In all cases, the virus peak was very sharp when high salt-treatedfeedstocks were used, with smaller and far broader peaks evident in theabsence of this treatment. An illustrative example of a typical sucrosegradient profile, with and without treatment is given in FIG. 1.

Example 5 High-Salt Treatment Greatly Increases Virus Yield in SucroseGradient-Purified Influenza B Virus

Samples of influenza B virus allantoic fluid were treated as in theprevious example for influenza A. Control samples were again clarifiedusing a coarse glass fibre filter. Each of the allantoic preparationswas fractionated on 6 mL sucrose step gradients gradients to achieve a17:1 loading ratio.

TABLE 7 Flu B/Hong Kong Gradient Loading Experiment Salt Treated HAUControl HAU Recovery Ratio 1 333,312 123,328 3:1 2 780,544 211,968 4:1 3727,298 108,032 7:1

Example 6 High-Salt Treatment Does Not Degrade Virus Infectious Titre

Gradient-purified influenza preparations with/without high-salttreatment were assayed by TCID₅₀ to assess the effect of treatment onvirus infectivity. Virus preparations were aliquoted, and one aliquot ofeach was mixed 1:1 with 3 M NaCl solution. Samples were incubated on icefor 1 hour, then clarified by centrifugation at 6,000 RPM for 5 minutes(Eppendorf Microcentrifuge). Supernatant was serially diluted ininfection medium and applied to MDCK cells in 96 well assay plates. CPEand/or HA status of each well was used to score presence of infection.The method of Reed and Muench (Amer. Jour. Hygiene, 27: 493-497, 1938)was used to calculate infectious titres.

TABLE 8 Infectious Titre Virus No Treatment High Salt FluA/Victoria/3/75 1.38 × 10⁸ PFU/mL 1.33 × 10⁸ PFU/mL Flu A/PR/8/34 2.18 ×10⁴ PFU/mL 2.18 × 10⁴ PFU/mL Flu A/2/Japan/305/57 9.20 × 10⁶ PFU/mL 5.17× 10⁶ PFU/mL Flu A/Hong Kong/8/68 2.18 × 10⁹ PFU/mL 1.38 × 10⁹ PFU/mLFlu A/X-31/Aichi/68 2.64 × 10⁸ PFU/mL 4.35 × 10⁷ PFU/mL Flu B/Lee/402.18 × 10⁴ PFU/mL 2.18 × 10⁴ PFU/mL

Table 8 indicates that high-salt treatment did not adversely affect thelive titre of the virus strains. Thus, high-salt treatment may beapplied to allantoic or other viral feedstocks without destruction ofvirus particles.

Example 7 Influenza Recovery Data From HA Assays, Infectious Titres andImmunoassays All Correlate

Fractions of an influenza A/B pool, retrieved after sucrose gradientpurification and titred by HA assay, were subjected to an opticalimmunoassay (OIA, Thermo BioStar).

TABLE 9 Fraction Number HA End-point  9 32,768 10 131,072 11 524,288 121,048,576

Samples of each gradient fraction were diluted 1:10, 1:100, and 1:400with PBS, then 100 μl aliquots were applied to BioStar sample tubescontaining disruption agent. Assays were performed according to theBiostar kit instructions, and color intensity was graded (1-7) against ascale provided in the kit.

TABLE 10 Gradient Pre-Dilution Fraction 1:10 1:100 1:400  9 5+ 4+ 1+ 107+ 6+ 2+ 11 7+ 6+ 4+ 12 Out of Range 6+ 4+

Hemagglutination assays are virus/strain sensitive, but are all relatedto the ratio of virus particles to red blood cells. As such, HA reflectsthe number of virus particles in a preparation. Thermo BioStar's Flu OIAtest is a rapid immunoassay which reports the presence of influenzanucleoprotein, therefore inferring the presence of virus particles. OIAcolor intensity results correlated with the determined HA titres.

Fractions of an influenza A/B pool, retrieved after sucrose gradientpurification and titred by HA assay, were also subjected to TCID₅₀assay.

TABLE 11 Comparison of HA titre and infectious titre in select gradientfractions TCID Fraction HA Titre HA Ratio TCID Titre Ratio 5 256 1 1.94× 10⁵ 1 14 524288 2048 3.73 × 10⁷ 192 17 16384 64 1.50 × 10⁷ 77

There was a correlation between the assays, in that highest HA titrecorresponded to highest infectious titre, and lowest HA titre similarlyhad the lowest infectious titre. To facilitate comparison, a ratio of HAtitre and of TCID₅₀ titre were calculated, relative to the lowest scoremeasured.

Example 8 Treated Influenza Virus Remains Intact

Preliminary transmission electron microscopy (TEM) studies wereperformed comparing peak gradient fractions of salt-treated versuscontrol preparations of influenza. Formvar-coated copper TEM specimengrids were floated on droplets (50 μl) of Influenza A/New Caledoniagradient fractions, and the samples adsorbed for 15 minutes at roomtemperature. Grids were washed twice with PBS, fixed with 0.1%glutaraldehyde in PBS (5 minutes), then washed twice using 0.2μm-filtered WFI water and negative stained for 1 minute with 2%phosphotungstic acid. Specimens were air dried, then examined on aHitachi H-7000 Transmission Electron Microscope using an acceleratingvoltage of 75 kV. Images were captured electronically in a 12-bitgrayscale compressed TIF format using a Hamamatsu ORCA HR CCD camera(AMT XR-60 imaging system).

Virus particles were observed in preparations that had been treated withhigh salt prior to gradient fractionation, and appeared to bemorphologically intact and the same as untreated controls. Virions hadan intact envelope, which negative stain failed to penetrate, andprominent surface spikes. Spherical and pleomorphic virion forms wereobserved in both treated and control preparations.

Virions in Control preparations were often associated with debris and/orwere attached to a mesh-like fibrous contaminate, which by negativestain seemed to condense around or encapsulate the virions. In contrast,virions in the salt-treated preparations were predominantlymonodisperse. Moreover, in the salt-treated preparations, the nature ofthe contaminating fibrous matrix appeared to have changed and there wasno obvious association of the fibrous matrix with the virions.

Example 9 Gradient Peak Analysis by Refractometer Indicates VirusDensity is Not Altered by Treatment

Gradient fractions characterized by HA analysis were concomitantlyanalyzed by optical refractometer to determine density corresponding toHA peak activity. A Misco Palm Abbe model PA200 was used to measurerefractive index for each gradient fraction, which were in turnconverted to density values using standard look-up tables for sucrosesolutions.

TABLE 12 Refractive index corresponding to peak HA activity for sucrosegradient fractionation of Influenza A/Texas allantoic pools. RefractiveIndex of Virus Peak Experiment Salt-Treated Control Run A (FPD4.016)1.4020–1.4072 1.4038–1.4089 Run B (FPD4.018) 1.4056–1.4078 1.4039–1.4068Run C (FPD4.020) 1.4059–1.4081 1.4074–1.4096

TABLE 13 Refractive index corresponding to peak HA activity for sucrosegradient fractionation of Influenza B/Hong Kong allantoic pools.Refractive Index of Virus Peak Experiment Salt-Treated Control Run A(FPD4.016) 1.4068 1.4069–1.4093 Run B (FPD4.018) 1.4032–1.4086 1.4063Run C (FPD4.020) 1.4079–1.4096 1.4079

Refractive index data for Influenza A/Texas and Influenza B/Hong Kongsucrose gradients are summarized in Table 12 and Table 13. For eachexperimental run and for both test viruses, there was close correlationof HA peak fraction density of salt-treated versus control allantoicspecimens.

Example 10 Diluting Virus-Containing Allantoic Fluid Prior to TreatmentVirus Yield

Each allantoic virus preparation (Influenza B/Yamanashi and InfluenzaA/Moscow) included four test samples (100 mL each) for gradientpurification. Controls were unfiltered (Series A) or clarified through aglass fiber depth-type filter (Series B). Treated samples wereprediluted by addition of 0.5 volume PBS, bringing the sample volume to150 mL, then an equal volume (150 mL) of 20×PBS was added and incubated1 hour overnight at 4° C. These treated samples were re-concentrated to100 mL using a 500 kda-cutoff hollow fiber filter, and either notclarified (Series C) or clarified by low trifugation (Series D). Allwere subjected to sucrose gradient purification, fractionated, andassessed by HA assay.

The results are summarized in Tables 14 and 15.

TABLE 14 Influenza B/Yamanashi Gradient [A]: Gradient [D]: FilteredGradient [B]: Gradient [C]: Salt-Treated Control Control Salt-Treatedand Filtered Fraction Allantoic Allantoic Allantoic Allantoic NumberFluid Fluid Fluid Fluid 1 5,120 327,680 20,480 81,920 2 10,240 163,840163,840 81,920 3 163,840 163,840 335,544,320 81,920 4 655,360 655,3605,242,880 2,621,440 5 655,360 327,680 335,544,320 10,485,760 6 655,360163,840 41,943,040 2,621,440 7 655,360 81,920 327,680 163,840 8 327,68081,920 40,960 40,960 9 81,920 40,960 40,960 20,480 10 81,920 20,48020,480 20,480 11 40,960 10,240 10,240 10,240 12 40,960 5,120 10,24010,240 Total HA 3,374,080 2,042,880 718,909,440 16,240,640

TABLE 15 Influenza A/Moscow Gradient [A]: Gradient [D]: FilteredGradient [B]: Gradient [C]: Salt-Treated Control Control Salt-Treatedand Filtered Fraction Allantoic Allantoic Allantoic Allantoic NumberFluid Fluid Fluid Fluid 1 320 320 10,240 10,240 2 640 640 10,240 20,4803 1,280 1,280 20,480 40,960 4 2,560 2,560 81,920 81,920 5 1,280 2,560327,680 81,920 6 1,280 1,280 1,310,720 81,920 7 320 320 5,242,880 20,4808 20 160 2,621,440 5,120 9 80 160 40,960 5,120 10 40 160 20,480 2,560 1120 80 10,240 2,560 12 20 40 5,120 2,560 Total HA 7,860 9,560 9,702,400355,840

Control samples yielded approximately the same amount of HA units foreach test virus, regardless of whether they were clarified byfiltration. The controls which were not filtered prior to gradientseparation had large pellets.

In contrast, the diluted then salt-treated preparations yielded muchhigher HA titres than the non-treated controls. Clarification was notnecessary for virus banding, and give the highest yields. Virus removedby clarification following the salt treatment was not optimallyreclaimed, hence yields are lower relative to the non-clarified samples.However, significant yield improvements relative to controls were stillachieved by pre-dilution and salt treatment irrespective ofclarification prior to gradient separation. Table 14 indicates that, forInfluenza B/Yamanashi, yields relative to the filtered control (SeriesA) were increased 213-fold in the test sample that lacked clarification(Series C) and 5-fold in the clarified test sample (Series D),respectively. Table 15 indicates that, for Influenza A/Moscow, yieldsrelative to the filtered control were increased 1,234-fold in the testsample that lacked clarification and 45-fold in the clarified testsample, respectively.

It will be apparent from the prior illustrative examples of practice ofthe invention that recovery of virus from allantoic fluid through use ofelevated salt treatment can readily be optimized by adjustments involume/salt content so that the salt concentration will not be so highas to precipitate allantoic fluid proteins (and virus associatedtherewith) nor so low as to fail to optimally function in disassociationof virus from allantoic fluid debris. Such optimization procedures arereadily carried out through making a preliminary analysis of the pooledallantoic fluid to be subject to salt treatment and adjusting the volumeof the pooled fluid based on these initial tests. In this manner,batch-to-batch, and possibly even strain-to-strain, variations inallantoic fluid proteins are accounted for.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims. Likewise, while the aboveillustrative examples all relate to improvement of yields from allantoicfluids in which various strains of influenza A and B virus have beengrown, the methods of the invention are readily applied to otherenveloped viruses typically grown in the allantoic fluid ofvirus-infected chick embryos. Indeed, the enhanced recoveries associatedwith practice of the present invention are likely to render use ofegg-based viral growth a method of choice for viruses now grown inmammalian cell culture provided standard adaptions of virus to suchgrowth are performed.

The references cited herein throughout, to the extent that they provideexemplary procedural or other details supplementary to those set forthherein, are all specifically incorporated herein by reference.

1. A process for recovering virus from debris-containing allantoic fluidof virus-infected chick embryos, comprising the steps of: (a) adding oneor more salts to the allantoic fluid to generate a total saltconcentration therein of greater than 0.5 M thereby dissociating virusfrom the debris; and (b) recovering virus dissociated from debris andsolubilized in the allantoic fluid.
 2. The process of claim 1, whereinthe salt is added to the allantoic fluid prior to removing the allantoicfluid from an egg.
 3. The process of claim 1, wherein recovery step (b)comprises clarifying the allantoic fluid of step (a) by centrifugationto form a debris-containing pellet and a virus-containing supernatant.4. The process of claim 3, further comprising dissociating virus fromthe pellet by suspending the pellet into a solution having a total saltconcentration of 0.5 M or greater and recovering virus from theresulting suspension.
 5. The process of claim 1, wherein recovery step(b) comprises clarifying the allantoic fluid of step (a) by filtrationto form a virus-containing filtrate and a debris-containing filterretentate.
 6. The process of claim 1, wherein step (b) comprises sucrosedensity centrifugation of allantoic fluid of step a) to localize viruswithin the density gradient.
 7. The process of claim 1, wherein thevirus is an enveloped virus.
 8. The process of claim 7, wherein theenveloped virus comprises an RNA genome.
 9. The process of claim 8,wherein the enveloped virus is a member of a virus family selected fromthe group consisting of orthomyxoviridae, paramyxoviridae, flaviviridae,togaviridae, rhabdoviridae, and coronaviridae.
 10. The process of claim9, wherein the virus is an influenza virus.
 11. The process of claim 10,wherein the virus is an Influenza A virus.
 12. The process of claim 10,wherein the virus is an Influenza B virus.
 13. The process of claim 1,comprising diluting the allantoic fluid prior to the addition of saidone or more salts.
 14. The process of claim 11, wherein the virus isMoscow strain of Influenza A.
 15. The process of claim 1, wherein atotal salt concentration from 1.0 M to 3.5 M is generated in step (a).16. The process of claim 15, wherein said one or more salts comprisesodium chloride.
 17. The process of claim 1, wherein said one or moresalts are contained within a phosphate buffered solution.
 18. Theprocess of claim 1, wherein the pH of the allantoic fluid is adjusted toor maintained in the range of pH 3 to
 10. 19. In a process for recoveryof virus from debris-containing allantoic fluid of virus-infected chickembryos wherein the allantoic fluid is subjected to clarification toform a clarified liquid fraction and a debris-containing fraction, theimprovement comprising extracting virus from both the clarified liquidfraction and the debris-containing fraction.
 20. The process of claim19, wherein said extracting step comprising: (a) dissociating virusassociated with the debris-containing fraction into a suspension with asolution of one or more salts having a non-isotonic total saltconcentration therein; and (b) recovering dissociated virus from thesuspension.
 21. The process of claim 20, wherein said extracting stepcomprises: (a) dissociating virus associated with the debris-containingfraction into a suspension with a solution of one or more salts having atotal salt concentration therein of 0.5 M or greater; and (b) recoveringdissociated virus from the suspension.
 22. The process of claim 21,wherein said clarification comprises centrifugation and saiddebris-containing fraction comprises a centrifugation pellet.
 23. Theprocess of claim 21, wherein said clarification comprises filtration andsaid debris-containing fraction comprises a filter retentate.
 24. Theprocess of claim 21, further comprising the step of recovering virusfrom the suspension by clarifying the suspension to form a secondclarified liquid and a second debris-containing fraction.
 25. Theprocess of claim 24, further comprising the step of recovering virusfrom the second clarified fluid by localization on a sucrose densitygradient.
 26. The process of claim 21, wherein the virus is an envelopedvirus.
 27. The process of claim 26, wherein the enveloped viruscomprises an RNA genome.
 28. The process of claim 27, wherein theenveloped virus is a member of a virus family selected from the groupconsisting of orthomyxoviridae, paramyxoviridae, flaviviridae,togaviridae, rhabdoviridae, and coronaviridae.
 29. The process of claim28, wherein the virus is an Influenza virus.
 30. The process of claim29, wherein the virus is an Influenza A virus.
 31. The process of claim29, wherein the virus is an Influenza B virus.
 32. The process of claim21, comprising diluting the allantoic fluid prior to the addition ofsaid one or more salts.
 33. The process of claim 30, wherein the virusis Moscow strain of Influenza A.
 34. The process of claim 21, whereinsaid total salt concentration in said suspension is from 1.0 M to 3.5 M.35. The process of claim 34, wherein said one or more salts comprisesodium chloride.
 36. The process of claim 21, wherein the pH of theallantoic fluid is adjusted to or maintained in the range of pH 3 to 10.37. A process for recovering influenza virus from debris-containingallantoic fluid of virus-infected chick embryos comprising the steps of:a) adding one or more salts to the allantoic fluid to generate a totalsalt concentration therein of 1.0 M or greater thereby dissociatingvirus from the debris; b) clarifying the allantoic fluid bycentrifugation or filtration; c) subjecting clarified allantoic fluid tosucrose density gradient separation to localize virus within the densitygradient; and d) isolating localized virus from the gradient.
 38. Theprocess according to claim 37, wherein the allantoic fluid of step a)has a pH adjusted to or maintained in the range pH 3.0 to pH 6.8. 39.The process according to claim 37, wherein the allantoic fluid of stepa) has a pH adjusted to or maintained in the range pH 6.8 to pH 9.8. 40.The process according to claim 37 further comprising suspending thepellet resulting from centrifugation or retentate resulting fromfiltration in a salt solution providing a total salt concentration of1.0 M or greater.